Introduction
Trimethylamine is ubiquitous in all major kingdoms of life, performing a
notable physiological and pathophysiological role (Loo, Chan, Nicholson,
& Holmes, 2022). Trimethylamine plasma concentrations directly
correlate to several human diseases, such as chronic kidney disease
(CKD), cardiovascular diseases, etc. (Yanget al. , 2019). The primary source of trimethylamine and TMAO in
humans are various precursors such as carnitine, choline, betaine, etc.
(Koeth et al. , 2013). In the human gut,
microbes convert dietary precursors into trimethylamine. This
trimethylamine gets converted into TMAO by FMOs
(Falony, Vieira-Silva, & Raes, 2015) in the
liver for excretion. Trimethylaminuria is an accumulation of
trimethylamine that causes a fish-like odor among people who lack a
functional FMO.
Trimethylamine and TMAO are significant carbon and nitrogen sources for
methylotrophs (Colby & Zatman, 1973; Loo et al. , 2022).
Methylotrophic bacteria are primarily found in the marine environment
and possess FMOs. The FMO, trimethylamine monooxygenase (Tmm )
that converts trimethylamine into TMAO (Liet al. , 2017) is an NADPH-dependent mono-oxygenase
(Chen, Patel, Crombie, Scrivens, & Murrell,
2011). This enzyme is assayed by monitoring NADPH concentration. Most
of the reported assays monitor NADPH conversion into
NADP+. Even techniques like the isothermal
calorimetry (Catucci, Sadeghi, & Gilardi, 2019)
and detection of NADPH (Dixit & Roche, 1984)
have limitations. Although sensitive, these methods can’t be used with
crude samples because other enzymes also use NADPH
(Spaans, Weusthuis, van der Oost, & Kengen,
2015). Hence it is normal to subtract background signals to obtain
enzymatic activity measurements. The stability of NADPH is also
sensitive to low pH and high temperatures (Wu,
Wu, & Knight, 1986). These limitations make NADPH-dependent assays
inaccurate.
Quantifying TMAO and trimethylamine directly in a sample where their
concentrations change is quite challenging. Given that TMAO and
trimethylamine are simultaneously found in clinical samples
(Gątarek & Kałużna-Czaplińska, 2021a), a
precise quantification of TMAO requires trimethylamine separation
(Awwad, Geisel, & Obeid, 2016a). Existing
methodologies take long durations to quantify TMAO precisely as
extraction and separation techniques result in sample loss. The amount
of TMAO or trimethylamine present in biological samples like urine and
blood plasma usually lies in the range of nM - µM
(Gątarek & Kałużna-Czaplińska, 2021b). GC-MS
detection conditions often convert TMAO to trimethylamine
(daCosta, Vrbanac, & Zeisel, 1990). Sample
pre-treatment is, therefore, almost always needed to eliminate
trimethylamine. Such procedures require column derivatization in which
solvent plays a significant role as these reactions do not proceed in an
aqueous media (Wang et al. , 2014).
Various other methods use NMR (He et al., 2021a), UHPLC (Awwad, Geisel,
& Obeid, 2016b; Ocque, Stubbs, & Nolin, 2015), LCMS/MS (Hefni,
Bergström, Lennqvist, Fagerström, & Witthöft, 2021), and HPLC (Lang et
al., 1998), but these are not cost-effective.